A fourth model, OCIP19, also showed growth inhibition, but this effect did not reach statistical significance, whereas there was no significant growth inhibition in OCIP18. By twice western blot, phosphorylated Akt and S6 were readily detected in these tumours excised 2h after the final dose of NVP-BEZ235 (Figure 7), although it should be noted that the sample processing was considerably longer than that for the acute time course experiment shown in Figure 4, which might have affected these results. Compound concentrations in the tumour tissue 2h after last treatment were in average of 1.11, 1.30, 0.81, and 1.16nmolg?1, for OCIP16, 18, 17, and 21 respectively. Hence the lack of efficacy observed in the model OCIP18 cannot be accounted for by a lack of exposure.

NVP-BEZ235 did not show accumulation over time in all the four tumour models tested, arguing that the efficacy is likely due to similar effects observed after a single-dose administration. Figure 5 Animal weights during chronic administration of NVP-BEZ235 or vehicle control. Figure 6 Tumour weights at the completion of chronic dose administration. Horizontal lines indicate the mean value for each group. Figure 7 Western blots of tumour lysates obtained 2h after the final dose of NVP-BEZ235 in the chronic dosing groups of animals, probed for Ser473 Akt and Ser235/236 and Ser240/244 S6 ribosomal protein, with actin loading controls. Owing to their small …

Effects of NVP-BEZ235 on proliferation, apoptosis, and angiogenesis markers To investigate the mechanisms of tumour growth inhibition by NVP-BEZ235, the tumours were labelled for cleaved caspase 3 and the proliferation markers cyclin D1 and p27 by immunohistochemistry, and the staining intensities were measured using semi-automated image analysis protocols. Cleaved caspase 3 showed low levels of staining relative to that previously observed by us in xenografts established from cell lines, and there was no significant increase in staining in any of the primary xenograft groups treated with NVP-BEZ235. There was a trend towards reduced labelling for cyclin D1 in the NVP-BEZ235-treated groups (Figure 8), and this was statistically significant in OCIP17, 19, and 21, whereas in the non-responsive OCIP18 model there was a non-significant increase in cyclin D1 in the NVP-BEZ235-treated animals. No significant effects on p27 were seen in any of the five primary xenograft models. Examination of newly formed blood vessels using CD31 staining showed that these were substantially Entinostat present in the surrounding fibrovascular stroma, rather than invading into tumour masses. We tested several image analysis routines based on the number of CD31-stained blood vessels, their size, and distribution within tumour tissue and stroma.